This research aims to elucidate reaction intermediates of NO-reductase activity in diiron proteins, with the ultimate goal of understanding how the metal clusters catalyze this reaction. Our studies will focus on three enzymes: 1) denitrifying NO reductases cNOR from Paracoccus denitrificans and qCuANOR from Bacillus azotoformans, 2) the [heme-copper] ba3 terminal oxidase from Thermus thermophilus, and 3) detoxifying NO reductase flavoprotein A (FprA) from Moorella thermoacetica. A better understanding of microbial NO reductases is highly desirable considering that these enzymatic reactions provide a resistance to the mammalian immune response. Although crystal structures exist for some of these enzymes, the structure and reactivity of their NO-complexes are not known. Diiron proteins participate in both detoxifying and denitrifying NO reductase reactions and are thought to react with NO to form [FeNO]2 intermediates. Alternative mechanistic models are considered and tested in this proposal. Resonance Raman and FTIR spectroscopies have the unique capability to identify nitrosyl intermediates and to define their NO-binding geometries with regard to the two metal ions. Novel rapid freeze-quench (RFQ) instrumentation that can trap intermediates within a sub-ms timescale provides new opportunities to characterize reaction intermediates that were previously inaccessible to spectroscopic methods. FTIR spectroscopy, in conjunction with low temperature photolysis of N3~ and CO, acting as NO surrogates, offers insight into binding geometries, potential hydrogen bond interactions and proton transfers relevant to these NO reductase mechanisms.